Cultivation Of Hyperthermophile Pyrococcus Furiosus, And Expression Of Its Cloned α-Amylase Gene

α-Amylases are among the most important commercial enzymes,having wide applications in starch-processing, brewing, alchohol production, textile, and other industries.Since starch starts being soluble only at 100℃ and above, the majority of industrial applications of α-amylasesrequire their use at temperatures of up to 110℃.The extracellular a-amylase from the hyperthermophilic archaeon Pyrococcus furiosus, PFA, is a promising candidate in starch processing, since it is optimally active at 100℃ and pH5.5-6.0 and does not require Ca2+ for activity or thermo- stability.The hyperthermophilic archaeon Pyrococcus furiosus was cultivated in an oxygen-free Schott bottle integrated with a thermometer, tubes gassing with nitrogen at 98 ℃. For massive culture,a10-liter high-pressure reactor was employed to perform the fermentation under absolutely anaerobic conditions for 52 hours. A typical yield of cells was 2.4g(wet weight), harvested from a working volume of 8 liters.Two oligonucleotides were synthesized according to the sequence of P. furiosus extracellular a-amylase gene amyA through the GenBank and used as primers for PCR with P. Juriosus genomicDNA as the template. The PCR product was inserted to plasmids pSE380 and then pJL3,yielding recombinants pSE-amyA, pJL-amyA. The recombinant vectors pSE-amyA, pJL-amyA were transformed into E.coli DH5α, Top10 and BL21(DE3). E.coli cells carrying these plasmids weregrown and induced with IPTG at 29℃ ,37℃ respectively. After induction,the a-amylase activity assay was carried out, showing the highest activity was 1.5U/ml (under 29 ℃ induction). Starch-containing SDS-PAGE also indicated the presence of active PFA. There is no reports on this a-amylase in China.